Cultures of Trypanosoma ambystomae were initiated and maintained in hy
po-osmotic biphasic blood-agar medium. Epimastigotes and trypomastigot
es occurred in culture and although they were similar in size, epimast
igotes predominated with about 70% of all stages. Epimastigotes divide
d by equal binary fission and occasionally formed rosettes. Cultured t
rypomastigotes did not divide. Cultures isolated 118 days earlier were
infective to all 3 laboratory-reared Ambystoma jeffersonianum and to
2 of 3 Ambystoma mexicanum; the latter is a new host record. Trypanoso
mes were morphometrically similar in both experimental hosts. Specimen
s from 20-day-old experimental infections were smaller than those from
natural infections. Dividing bloodstream forms were detected in exper
imental infections. Standard culture conditions should be adopted if t
he morphology of culture forms is to have taxonomic significance.